Compositions and methods for preventing and treating heterotopic ossification and pathologic calcification

ABSTRACT

The present invention is directed to compositions and methods for the prevention or treatment of treatment of heterotopic ossification, vascular calcification, or pathologic calcification.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 15/702,758,filed on Sep. 13, 2017, which claims the benefit of U.S. provisionalapplication 62/395,374, filed on Sep. 15, 2016. The contents of theseprior applications is incorporated by reference herein in theirentirety.

FIELD OF THE INVENTION

The present invention is directed to methods of preventing or treatingheterotopic ossification and other forms of pathologic calcification inpatients by administering drug combinations containing: a) an antagonistof the Hedgehog signaling pathway; b) vitamin D, cholecalciferol or avitamin D analog; and/or c) a statin. It also includes pharmaceuticalcompositions administered to patients in the performance of thesemethods. The invention is also directed to methods of treatment orprevention which utilize vitamin D, cholecalciferol or a vitamin Danalog in the absence of an Hh pathway antagonist or a statin; and whichuse a statin in the absence of an Hh pathway antagonist, vitamin D,cholecalciferol or a vitamin D analog.

BACKGROUND OF THE INVENTION

Heterotopic ossification (HO) is characterized by the formation ofectopic bone in soft tissues, such as the fibrous tissue adjacent tojoints. It is most commonly associated with trauma, such as spinal cordinjury, brain injuries, head injuries, burns, fractures, musclecontusions, combat-related trauma and surgery (especially jointarthroplasty). In addition, HO may occur in patients that are onneuromuscular blockade to manage adult respiratory distress syndrome andin patients with non-traumatic myelopathies. In rare cases, HO maypresent as part of a hereditary disease and is sometimes associated withlower motor neuron disorders. The consequences of HO may include, interalia, joint contracture and ankylosis, pain, spasticity, swelling,fever, neurovascular compression, pressure ulcers, and significantdisability. Lesions range from small, clinically insignificant foci tomassive deposits throughout the body.

NSAIDs, such as indomethacin, can be effective in the prevention of HOif treatment is started early and preoperative radiation may be used toprevent HO after total hip or knee arthroplasty. Combined postoperativeradiotherapy and indomethacin has also been suggested to be effective atpreventing HO. Unfortunately both radiation therapy and NSAID treatmenthave significant limitations and may result in serious side effects (seeBaird, et al., J. Ortho. Surg. Res. 4:12 (2009)).

There are currently no generally ffective treatments for ectopic boneformation due to genetic diseases. Areas of well-circumscribed HO cansometimes be surgically removed with successful long-term results butresection of diffuse lesions usually leads to recurrences orcomplications. Successful functional repositioning of a joint after thedevelopment of a contracture from HO may also occasionally be possible.However, treatment options are very limited and these diseases aregenerally severely disabling.

Recent analyses have suggested that the cellular origin for ectopic boneformation may be mesenchymal progenitor cells. The differentiation ofthese cells into osteogenic lineages is induced by a pathologicalmicroenvironment in soft tissues outside the skeletal tissue, whichincludes inflammation. Recent reports suggest that drugs that act asantagonists of the Hedgehog signaling pathway (which is essential forproper embryonic development and is believed to play a role in thedevelopment of some cancers) may be effectively used to prevent or treatHO as well as other pathological conditions characterized by ectopiccalcification (see e.g., US 2014/0220154). Further development oftreatment methods using Hh pathway antagonists and other agents isclearly warranted.

SUMMARY OF THE INVENTION

The present invention is based on the concept that vitamin D,cholecalciferol, vitamin D analogs; and statins can be used to inhibitosteogenesis in mesenchymal stem cells and to prevent or treatheterotopic ossification, vascular calcification, or pathologiccalcification in a patient. It is further based on the concept thatcombinations of these agents, and combinations of these agents togetherwith Hedgehog (Hh) pathway antagonists, can produce a greater effect infunctional assays of osteogenesis prevention than agents used alone.Effects may include changes in gene expression and a reduction inalkaline phosphatase (AP) activity, a sign of prevented celldifferentiation and HO aversion. As AP gene expression correlates withosteogenesis, the drugs and drug combinations discussed herein arebelieved to be effective in decreasing the cascade leading to HO and toact in a dose-dependent and synergistic manner. This may allow one tooptimize the safety profile of a treatment by minimizing the individualagent's quantity and thus reducing toxicity associated with high dosesof compounds. Treatment should be especially effective in those patientsin which HO is associated with aberrant Hh signaling activity.

In its first aspect, the invention is directed to a method forinhibiting osteogenesis in mesenchymal stem cells, by contacting thecells with:

-   -   a) a combination of a Hedgehog (Hh) pathway antagonist together        with:        -   i) vitamin D, cholecalciferol or a vitamin D analog; or        -   ii) a statin;    -   b) a combination of:        -   i) vitamin D, cholecalciferol or a vitamin D analog; and        -   ii) a statin; or    -   c) a combination of:        -   i) a Hh pathway antagonist;        -   ii) vitamin D, cholecalciferol or a vitamin D analog; and        -   iii) a statin.

In all of the combinations, the compounds are administered to cells in aco-timely manner, preferably concomitantly and more preferably,essentially simultaneously. The dosage of each compound should besufficient to make the combination effective at inhibiting osteogenesis.Also, the combination in paragraphs a), b) or c) should produce agreater degree of inhibition than any components of the combination canproduce when used in the absence of all of the components. The mostpreferred of the combinations contains all three of: i) an Hh pathwayantagonist; ii) vitamin D, cholecalciferol or a vitamin D analog; andiii) a statin.

The invention is also directed to a method for preventing or treatingheterotopic ossification, vascular calcification, or other pathologiccalcification in a patient, by administering any of the combinations setforth above. These agents should be administered in a co-timely manner(i.e., a therapeutically effective amount of a first drug should bepresent in the patient when the second drug is given and, if a thirddrug is given, then both a therapeutically effective amount of the firstdrug and a therapeutically effective amount of the second drug should bepresent when the third drug is administered). Preferably the drugs areadministered concomitantly (within one hour of one another) and stillmore preferably, essentially simultaneously (within five minutes of oneanother) or in a single unit dosage form. The dosage of the compoundsgiven to patients should be sufficient to make the combinationtherapeutically effective and, preferably the combination of drugsshould be more effective at preventing or treating heterotopicossification, vascular calcification, or other pathologic calcificationin a patient, than any single component of the combination whenadministered in the absence of the other components. The most preferredcombination has all three of: i) an Hh pathway antagonist; ii) vitaminD, cholecalciferol or a vitamin D analog; and iii) a statin.

This treatment may be used for patients with ectopic bone formation orcalcification of any origin, including spinal cord damage, traumaticinjuries, head and brain injuries, burns, bone fractures, muscleinjuries, and surgery. It should also be effective in patients withdiseases or conditions that predispose them to ectopic bone formation orcalcification, such as atherosclerosis or myocardial infarction, and ingenetic diseases such as osseous heteroplasia, fibrodysplasia ossificansprogressiva and Albright's hereditary osteodystrophy.

The dosage administered to a patient will vary depending on theparticular agents being administered, the route of administration andclinical factors unique to the individual being treated. As a guidehowever, it is expected that, when administration is by an oral ortransdermal route, patients will typically receive a combination of1-500 mg/day of an Hh pathway antagonist; 300-3000 IU/day of vitamin D,cholecalciferol or a vitamin D analog; and/or 1-500 mg/day of a statin.Similar dosages may also be used for systemic or local injection but, inthese instances, it may be possible to reduce the dosage somewhat (e.g.,by 20-80%). These dosages apply regardless of which combination isadministered to a patient.

An Hh pathway antagonist may be an inhibitor of mesenchymal stem cellsand, structurally, may take the form of a protein or peptide ligand thatbinds to the Sonic receptor and prevents activation, an antibody thatbinds to either Sonic, Desert or Indian or to the receptor for theseligands, or an siRNA that inhibits the expression of one or more genesactivated by the Hh pathway. Specific Hh pathway antagonists that may beused in compositions include: a) zerumbone epoxide; b) staurosporinone;c) 6-hydroxystauro-sporinone; d) arcyriaflavin C; e)5,6-dihyroxyarcyriaflavin A; f) physalin F; g) physalin B; h)cyclopamine; i) HPI-1, HPI-2; HPI-3; or HPI-4; j) arsenic trioxide(ATO); k) sodium arsenite; l) phenylarsine; m) GANT-58; n) GANT-61; o)zerumbone; and p) inhibitors of the expression of the genes Ptch1, Gli1or HIP. Of these the most preferred is arsenic trioxide (ATO)administered at a dose of 0.05 to 0.20 mg/kg/day, and more preferably at0.10 to 0.20 mg/kg/day. Preferred combinations also include vitamin D orcholecalciferol and a statin selected from the group consisting of:Atorvastatin; Fluvastatin; Pravastatin; Rosuvastatin; Simvastatin;Pitavastatin; Cerivastatin; Lovastatin; and Mevastatin.

Overall, a particularly preferred method of preventing or treatingheterotopic ossification, vascular calcification, or pathologiccalcification in a patient, includes the co-timely (and more preferablyconcomitant or essentially simultaneous) administration of: a) an Hhpathway antagonist selected from the group consisting of: zerumboneepoxide; staurosporinone; 6-hydroxystauro-sporinone; arcyriaflavin C;5,6-dihyroxyarcyriaflavin A; physalin F; physalin B; cyclopamine; HPI-1,HPI-2; HPI-3, or HPI-4; arsenic trioxide; sodium arsenite; phenylarsine;GANT-58; GANT-61; zerumbone; and inhibitors of the expression of thegenes Ptch1, Gli1 or HIP, with the most preferred being arsenictrioxide; b) vitamin D or cholecalciferol; and c) a statin selected fromthe group consisting of Atorvastatin; Fluvastatin; Pravastatin;Rosuvastatin; Simvastatin; Pitavastatin; Cerivastatin; Lovastatin; andMevastatin.

The dosage of each compound should be sufficient to make the overalltreatment therapeutically effective and preferably synergistic. In thecontext used herein, the term “synergistic” means that the effect of acombination of drugs is greater than the maximum effect that can beachieved when the drugs are used individually. For example, thecombination of ATO, cholecalciferol and levostatin would be actingsynergistically with respect to preventing heterotopic ossificationafter surgery if, compared to an untreated group, fewer people developedectopic ossification when given the combination then when administeredany one component of the combination alone. When used to treat existingectopic ossification, synergism may occur with respect to one or moresymptoms associated with ossification such as pain, swelling, range ofjoint motion etc. Synergism may also manifest itself in other ways, suchas the rapidity with which relief from a symptom is first experienced,or the duration of action.

In addition, the treatment methods described herein and the compositionsused to accomplish those methods, may exhibit a reduction in thefrequency or severity of one or more side effects relative to thatproduced by administering one of the components alone. Specific sideeffects may be reduced include weakness, fatigue, drowsiness, headache,loss of appetite, dry mouth, metallic taste, nausea, vomiting, liverdamage, kidney failure, rhabdomyolysis, pleural effusion, fever,palpitations, tachycardia, weight gain, dyspnea, leukocytosis, prolongedqt interval on ECG, and cardiac arrhythmia.

In another aspect, the invention is directed to a pharmaceuticalcomposition in unit dosage form, which includes any of the combinationsset forth above. These agents should be present in an amount such thatthey are therapeutically effective upon the administration of one ormore unit dosage forms to a patient. Although only two agents may bepresent, it is preferred that all three be included in a single unitdosage form.

For dosage forms designed for oral delivery (e.g., pills, tablets orcapsules) or for transdermal delivery (e.g., patches, gels, foams orointments), the Hh antagonist should typically be present in thecompositions in an amount of 1-500 mg; vitamin D, cholecalciferol or avitamin D analog should be present at 100-3000 IU; and the statin shouldbe present at 1-500 mg. The most preferred composition has ATO at 1-100mg; cholecalciferol at 100-3000 IU; and a statin selected fromAtorvastatin; Fluvastatin; Pravastatin; Rosuvastatin; Simvastatin;Pitavastatin; Cerivastatin; Lovastatin; and Mevastatin.

Dosage forms for injection are essentially the same as those for oraldelivery and comprise: an Hh antagonist at 1-500 mg; vitamin D,cholecalciferol or a vitamin D analog at 300-3000 IU; and a statin at1-500 mg. However somewhat lower amounts may also be possible. Thus, adosage forms designed for local or systemic injection (or forimplantation) may have an Hh antagonist present at 0.1-50 mg; vitamin Dor cholecalciferol present at 100-3000 IU or an equivalent amount of avitamin D analog; and a statin at 0.1-50 mg. Specifically preferredcomponents are the same as for oral compositions.

The Hh antagonist used in compositions may be an inhibitor ofmesenchymal stem cell differentiation and may take the form of proteinor peptide ligand that binds to the Sonic receptor and preventsactivation; an antibody that binds to either Sonic, Desert or Indian orto the receptor for these ligands; or an siRNA that inhibits theexpression of one or more genes activated by the Hh pathway. The mostpreferred compositions will be in the form of a pill, tablet or capsulefor oral administration or a patch, gel, foam or ointment for topicaladministration, and comprise: a) 1-500 mg of an antagonist of the Hhpathway; (b) 100-3000 IU of vitamin D or cholecalciferol; and c) 1-500mg of a statin. The Hh pathway antagonist may be any of those recitedherein but will most preferably be arsenic trioxide at 5-50 mg. The mostpreferred statin is selected from the group consisting of: Atorvastatin;Fluvastatin; Pravastatin; Rosuvastatin; Simvastatin; Pitavastatin;Cerivastatin; Lovastatin; and Mevastatin.

The invention also includes a method for manufacturing a sterilepharmaceutical formulation comprising a) arsenic trioxide; and b)vitamin D or cholecalciferol. This is accomplished by: (a) solubilizingarsenic trioxide in an aqueous solution at a pH greater than 10; (b)neutralizing the arsenic trioxide solution with an acid to a pH between8 and 10; (c) diluting the arsenic trioxide solution from step (b) in apharmaceutical carrier that stabilizes it and lowers the pH to about 7(e.g., from 6.8 to 7.2); (d) emulsifying the arsenic trioxide withvitamin D, cholecalciferol or a vitamin D analog; and (e) sterilizingthe pharmaceutical composition. If desired, one or more statins may besubstituted for the vitamin during emulsification or may be included asa third component.

All of the pharmaceutical compositions may be used in the prevention ortreatment of heterotopic ossification, vascular calcification, orpathologic calcification according to the methods and dosages describedherein.

The present invention is also directed to a method for inhibitingosteogenesis in mesenchymal stem cells and to a method for preventing ortreating heterotopic ossification, vascular calcification, or otherpathologic calcification in a patient, by administering vitamin D,cholecalciferol or a vitamin D analog in the absence of a Hh pathwayantagonist or a statin; and by administering a statin in the absence ofa Hh pathway antagonist or any of vitamin D, cholecalciferol or avitamin D analog. Specific statins that may be used include:Atorvastatin; Fluvastatin; Pravastatin; Rosuvastatin; Simvastatin;Pitavastatin; Cerivastatin; Lovastatin; and Mevastatin.

Treatment of patients may be for ectopic bone formation or calcificationof any origin, including spinal cord damage, traumatic injuries, headand brain injuries, burns, bone fractures, muscle injuries, and surgery.The agents should also be effective in patients with diseases orconditions that predispose them to ectopic bone formation orcalcification, such as atherosclerosis or myocardial infarction, and ingenetic diseases such as osseous heteroplasia, fibrodysplasia ossificansprogressiva and Albright's hereditary osteodystrophy.

In all cases, patients should receive a therapeutically effective amountof a drug. It is expected that, when administration is by an oral ortransdermal route, patients will typically receive 300-3000 IU/day ofvitamin D, cholecalciferol or a vitamin D analog; or 1-500 mg/day of astatin. Similar dosages may also be used for systemic or local injectionbut, typically they will be reduced, e.g., by 20-80%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Alkaline phosphatase assay of primary BMSC, (P1) isolated fromwild type mice: a) Control, no agent present; b) Arsenic trioxide (ATO),10 μM present during osteogenesis; c) Cholecalciferol 10 μM present inmedia during osteogenesis; d) Lovastatin 1 μM present in media duringosteogenesis; e) NP101 present during osteogenesis diluted (1:1) f)NP101 present during osteogenesis diluted (1:10).

FIG. 2: Alkaline phosphatase assay of BMSC isolated from wild type miceand primed for osteogenesis; a) Control, no agent present; b) ATO, 10 μMpresent during osteogenesis; c) Cholecalciferol 10 μM present in mediaduring osteogenesis; d) Lovastatin 1 μM present in media duringosteogenesis; e) NP101 present during osteogenesis diluted (1:1) f)NP101 present during osteogenesis diluted (1:10).

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

Administration: As used herein this term refers both to drugs given to apatient by health care personnel (e.g., a physician or nurse) as well toself administration, i.e., patients taking medication themselves.

Co-timely Administration: As used herein, “co-timely administration”means administration of a subsequent drug to prevent or treatheterotopic ossification, vascular calcification, or pathologiccalcification during the time when a previously administered, first drugis still present in an amount in a patient that is therapeuticallyeffective in combination with the second drug. Thus, drugs must be givenin close enough temporal proximity that they can have a cumulativeeffect. If a third drug is given, then this must be done while both thefirst and second drugs are present in therapeutically effective amountswhen in combination with the third.

Concomitant Administration: As used herein, the term “concomitantadministration” means that drugs are given within one hour of oneanother.

Essentially Simultaneous Administration: As used herein this phraserefers to drugs that are given in a single unit dosage form or which aregiven within 5 minutes, and preferably 2 minutes of one another

Heterotopic Ossification: Heterotopic ossification is the deposition ofbone at sites in the body where it does not belong. Unless otherwiseindicated, the term as used herein refers to bone formation at anabnormal site wherever that site happens to be and regardless of thecause.

Vascular calcification: Vascular calcification refers to the depositionof calcium in blood vessel structures and is often associated withatherosclerosis. (Bostrom, et al., J. Clin. Invest. 91:1800-09 (1993)).The consequences of calcification of blood vessels can be severe and maylead to congestive heart failure, aortic stenosis and weakened vasomotorresponses.

Pathologic Calcification: For the purposes herein, pathologiccalcification may be considered to be the deposition of calcium salts insoft tissue causing a hardening, but not bone formation. Thus, the termincludes vascular calcification but also includes calcification outsideof the vasculature.

Vitamin D, Cholecalciferol and Vitamin D Analogs: As used herein“cholecalciferol” refers specifically to vitamin D3 whereas the term“vitamin D” comprises all forms of vitamin D (including vitamin D2(ergocalciferol) and D3 (cholecalciferol)) and combinations of theseforms. Unless otherwise indicated, dosages or quantities recited referto the total combined amount of all forms of vitamin D administered to apatient or present in a composition. The term “vitamin D analog refersto any compound (other than a naturally occurring human form of vitaminD) which has vitamin D biological activity and especially any suchcompound that binds to, and activates the vitamin D receptor (i.e., thecalcitrol receptor). Such receptors may be found, for example in humanosteoblasts, hepatocytes or immune cells. Examples of vitamin D analogsinclude but are not limited to those in the following US patentreferences (all of which are incorporated by reference herein in theirentirety): (U.S. Pat. Nos. 7,985,744; 8,198,263; 7,659,421; 7,211,680;7,115,758; 7,112,579; 7,074,777; 6,538,145; 6,359,152; 6,277,837;6,124,276; 6,043,385; 6,013,814; 5,945,410; 5,756,733; 5,700,791;5,665,716; 5,446,035; 5,232,836; 4,891,364; 4,857,518 4,851,400).

Statins: Statins are recognized in the art as a distinct drug class thatact as inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMG-CoA reductase), an enzyme involved in the synthesis of cholesterol.Structurally, they are characterized by a dihydroxyheptanoic acid group(sometimes in the form of a lactone) which forms a structure resemblingHMG-CoA (the substrate of HMG-CoA reductase). This group is attached toa variety of ring systems (including aromatic, heterocyclic oraromatic-heterocyclic, unsubstituted or substituted, mono-, di- orpoly-cyclic ring systems). Specific examples of statins includeAtorvastatin; Fluvastatin; Pravastatin; Rosuvastatin; Simvastatin;Pitavastatin; Cerivastatin; Lovastatin; and Mevastatin. These specificcompounds and all compounds recognized in the art as being a member ofthe statin drug class are included within the scope of the invention.

Hedgehog pathway antagonists: The hedgehog signaling pathway is involvedin the differentiation of cells during embryonic development and alsoappears to play a role regulating adult stem cells. Inhibition of thispathway has been reported to decrease the proliferation andclonogenicity of human mesenchymal stem cells which are known to becapable of differentiating into, inter alia, osteoblasts (see Plaisant,et al., PLoS One: 6(2):e16798 (2011)). Any inhibitor of this pathwayidentified in the art as being effective in humans is within the scopeof the invention regardless of its mechanism of action. This includes:small molecules that block the binding of a hedgehog ligand (Desert,Indian or Sonic) to its receptor; antibodies that target either ligandor receptor; agents that block intracellular activation after receptorbinding; and agents that block gene expression such as siRNAs. Examplesof specific inhibitors include: a) zerumbone epoxide; b)staurosporinone; c) 6-hydroxystaurosporinone; d) arcyriaflavin C; e)5,6-dihyroxyarcyriaflavin A; f) physalin F; g) physalin B; h)cyclopamine; i) HPI-1, HPI-2; HPI-3; or HPI-4; j) arsenic trioxide(ATO); k) sodium arsenite; l) phenylarsine; m) GANT-58; n) GANT-61; o)zerumbone; and p) inhibitors of the expression of the genes Ptch1, Gli1or HIP. Examples of other Hh pathway antagonists that may be used in theinvention include, but are not limited, to those in the following USpatent references (all of which are incorporated by reference herein intheir entirety): U.S. Pat. Nos. 9,427,431; 9,409,871; 9,346,791;9,345,699; 9,321,761; 9,278,961; 9,216,964; 9,174,949; 9,173,869;9,149,527; 9,096,686; 9,073,835; 9,000,023; 8,835,648; 8,802,639;8,778,927; 8,759,367; 8,530,456; 8,486,400; 8,410,601; 8,273,747;8,101,610; 8,030,454; 7,741,298; 7,407,967; 6,683,108; and 6,291,516.

Therapeutically effective amount: The term “therapeutically effectiveamount” means a dosage of drug that provides the specificpharmacological response for which the drug is administered in asignificant number of subjects in need of such treatment. With respectto the therapeutic agents discussed herein, this would be a dose of eachactive drug which, in combination (or in the case of vitamin D,cholecalciferol, a vitamin D analog or a statin, when used alone or incombination) reduces the number of patients developing heterotopicossification (e.g., after arthroscopic surgery, spinal injury, trauma,head or brain injuries, bone fractures or burns) by at least 15%(preferably at least 30% and more preferably at least 50%) relative toclinically matched patients that are not treated. In patients that arediagnosed as having heterotopic ossification, a therapeuticallyeffective amount is a dosage sufficient to reduce the clinical symptomsassociated with the condition to a greater degree in at least 15% ofpatients receiving treatment (preferably at least 30% and morepreferably at least 50%) relative to clinically matched patients thatare not treated. This reduction in symptoms should occur in less thanone year after treatment is begun and generally in less than one month.In most cases, a reduction in symptoms should be seen in less than oneweek, and preferably within only a day or two, after treatment isinitiated. Clinical symptom improvement may take the form of a greaterreduction in pain, swelling, or pressure or a greater shrinkage of boneor calcium deposits. Note that reference to “specific pharmacologicalresponse for which the drug is administered in a significant number ofsubjects in need of such treatment” is a recognition that a“therapeutically effective amount,” administered to a particular subjectmay not be effective in that patient even though such dosage is deemedto be therapeutically effective by those skilled in the art.

Unit dosage form: The term “unit dosage form” is defined as a singledrug administration entity. By way of example, a single pill, tablet,capsule, dragee, or trochee, or a specified volume of solution forinjection would be a unit dosage form. It also applies to a singledosage of drug applied transdermally in a patch or as part of aspecified amount of gel, foam or ointment.

B. Compounds

Hedgehog inhibitors have been thoroughly described in the literature andhave generated interest as potential therapies for cancer. Vitamin D,cholecalciferol and vitamin D analogs and methods for obtaining thesecompounds are also described in the literature as are statins. Since allof these compounds are well known, one of skill in the art will be ableto either purchase them or make them using information in the literatureand standard laboratory methods.

C. Drug Formulation and Dosage Forms

The compounds described herein may be administered to patients in apharmaceutical composition comprising the compounds along with apharmaceutically acceptable carrier. The carrier may be any solvent,diluent, liquid or solid vehicle that is pharmaceutically acceptable andtypically used in formulating drugs. Guidance concerning the making ofpharmaceutical formulations can be obtained from standard works in theart (see, e.g., Remington's Pharmaceutical Sciences, 16^(th) edition, E.W. Martin, Easton, Pa. (1980)). In addition, pharmaceutical compositionsmay contain any of the excipients that are commonly used in the art.Examples of carriers or excipients that may be present include, but arenot limited to, sugars (e.g., lactose, glucose and sucrose); starches,such as corn starch or potato starch; cellulose and its derivatives(e.g., sodium carboxymethyl cellulose, ethyl cellulose, or celluloseacetate); malt; gelatin; talc; cocoa butter; oils (e.g., peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, orsoybean oil); glycols; buffering agents; saline; Ringer's solution;alcohols; lubricants; coloring agents; dispersing agents; coatingagents; flavoring agents; preservatives; and antioxidants.

Although dosage forms for oral delivery are preferred, the invention iscompatible with the delivery of compounds by any route known in the art,including peroral, internal, rectal, nasal, lingual, transdermal,intravenous, intra-arterial, intramuscular, intraperitoneal,intracutaneous and subcutaneous routes. Liquid dosage forms for oraladministration include, but are not limited to, pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, liquid dosage forms maycontain inert diluents commonly used in the art, such as, for example,water, or other solvents, solubilizing agents and emulsifiers, such asethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethyl formamide, oils, glycerol, alcohols, polyethylene glycols, andfatty acid esters.

Injectable preparations may be in the form of sterile, injectableaqueous or oleaginous suspensions, diluents or solvents that may be usedmay include 1,3-butanediol, water, Ringer's solution and isotonic salinesolutions. In addition, oils or fatty acids may be present.

The most preferred dosage forms are forms such as capsules, tablets andpills. In these dosage forms, the active compound will typically bemixed with at least one inert, pharmaceutically acceptable excipient orcarrier such as sodium citrate, or dicalcium phosphate and/or: fillersor extenders such as starches, lactose, sucrose, glucose, mannitol andsilicic acid; binders such as, for example, carboxymethyl cellulose,alginates, gelatin, polyvinylpyrrolidinone, and acacia, humectants suchas glycerol; disintegrating agents such as calcium carbonate, silicatesor sodium carbonate; solution retarding agents such as paraffin;absorption accelerators such as quaternary ammonium compound; wettingagents such as cetyl alcohol or glycerol monostearate; absorbents suchas kaolin; and lubricants, such as talc, magnesium stearate; sodiumlauryl sulphate, etc. In addition, dosage forms may include bufferingand flavoring agents.

D. Dosage

Pharmaceutical compositions will typically be given to a patient in oneor more unit dosage forms that alone or together provide a dose that istherapeutically effective. The exact amount of each active drug informulations will vary depending on the specific drugs chosen and theroute of administration. In general, the dosage of Hh inhibitor givenorally will be about 1-500 mg daily, but higher or lower dosages may beused by physicians depending on the response of individual patients.Similar dosages would apply to transdermal formulations including thosedelivered using a transdermal patch or those in the form of a gel, foamor ointment. In the most preferred embodiment, arsenic trioxide is usedas the Hh antagonist and is administered to patients at a dosage ofabout 0.15 mg/kg/day. If injected, the dosage may be essentially thesame as for oral administration but it may also be possible to reducethe dosage by 20 to 80% or more.

The dosage of statin will be between 1 and 500 mg (and usually 1 to 100mg), given orally or transdermally per day. Again, if injected this maybe reduced by 20 to 80% or more.

In oral and transdermal formulations, the amount of vitamin D orcholecalciferol will typically be about 300-3000 IU per day. If ananalog is used, an amount of drug equivalent to 300-3000 IU of D3 may beused initially and adjusted according to patient response. If injectedthis may be reduced by 20 to 80%.

E. Treatment Methods

Subjects receiving therapeutic agents according to the methods describedherein will fall into two categories. The first consists of individualsthat do not yet have abnormal bone formation or calcification but arepart of a group recognized as being prone to this occurring. Included inthis group are patients that have undergone surgery (particularlyarthroscopic surgery of a hip or other joint), and those that haveundergone traumatic injuries, fractures, wounds, head or brain injuriesand burns. The group also includes subjects with atherosclerosis, thathave had a myocardial infarction or that have a genetic diseaseassociated with ectopic bone formation or calcification. The objectivein these cases is to reduce the likelihood of HO or abnormalcalcification occurring. In general, these patients will receive dailydosages that continue until the attending physician is satisfied thatincreased risk has subsided. This may be anywhere from a few weeks up toseveral years. In the case where increased risk of heterotopicossification, vascular calcification, or pathologic calcification is dueto genetic factors or ongoing disease, administration may be continuedfor the life of the patient.

The second category of patients will be those that have been identifiedclinically as already suffering from heterotopic ossification, orpathologic calcification and for whom the objective is primarily totreat the existing condition. In general, these patients will beadministered compositions in the same manner as those in which theobjective is prevention but dosages and dosing schedules may be varieddepending on the degree to which a response is observed and may becombined with physical therapy or surgery.

In all cases, treatment may be altered by physicians based on clinicalfactors unique to individual patients.

EXAMPLES

This example, is concerned with the effect of certain combinations ofcompounds on mesenchymal stem cells and bone marrow stromal cellsosteogenic differentiation.

Methods

Mesenchymal stem cells isolation: Bone marrow stromal cells wereisolated from six to eight weeks old wild type mice. Femurs and tibiaswere dissected from surrounding tissues. The epiphyseal growth plateswere removed and the marrow was collected by flushing with modifiedessential medium (MEM) containing 100 U/ml Penicillin, 100 μg/mlstreptomycin and 10% fetal bovine serum with a 25 G needle. Single cellsuspensions were prepared by passing the cell clumps through an 18Gneedle followed by filtration through a 70-μm cell strainer.

Priming cells for osteogenseis: An aliquot of cells was diluted 1:1 with0.04% trypan blue in PBS and viable cells were plated at a density of2×10⁶ cells/cm² and in a 35-mm culture dish and exposed to recombinantmouse Prolactin (PRL) (1 ng/ml) and/or Lacritin 2(LCN2) (18 ng/ml) inAlpha MEM media with 20% FBS, L-Glutamine 2 mM and Pen-Strep (1×) forthree passages to prime cells for osteogenesis. After the third passage,50,000 bone marrow stromal cells per well were plated in a 24 well platein Alpha MEM 20% FBS, L-Glutamine (4 mM), PenStrep (1×) and MEMNon-Essential Amino Acids (1×).

After cells reached confluence, medium was replaced with osteogenicdifferentiation medium (OM). Osteogenic differentiation medium (OM)consists of: DMEM, 10% FBS, Pen-Strep (1×), L-Glutamine (2 mM), 10 mMbeta glycerol phosphate and 50 μg/ml ascorbic acid. Duplicate wells wereexposed to differentiation media without any agent (control), singleagent (Cholecalciferol, ATO or Lovastatin) or Nosto-101 combinationagents. Media replacement occurred every 2-3 days with freshdifferentiation medium (made each time during feeding) with or withoutsingle agent or Nosto-101.

Alkaline phosphatase assay: Cells were stained after 4 days exposure inOM differentiation medium for Alkaline Phosphatase (AP) assay. This is acolorimetric assay in which NBT (nitro-blue tetrazolium chloride) andBCIP (5-Bromo-4-chloro-3′-indolyphosphate p-toluidine salt) yieldsinsoluble purple precipitate when reacted with alkaline phosphataseenzyme.

After 4 days of culturing cells in OM media, cells were fixed with 4%paraformaldehyde and then washed once with PBS. One ml of One-stepNBT/BCIP staining solution (Thermo Fisher) was added per well and thecells were then incubated in the dark for 30 min to 1 h and checked forcolor development. The reaction was quickly stopped by removing NBT/BCIPand adding water.

ATO: Arsenic trioxide stock solution was prepared by placing 50 mg ofATO (Sigma) at the bottom of a 50 ml conical tube and dissolving it with1 ml of 1M NaOH. 48 ml of PBS was then added and 1N HCl was used toadjust the pH to 7.2

Cholecalciferol (Sigma) was dissolved in absolute ethanol in 10 mM stockconcentration. Working concentration used in cell culture experimentswas 1004.

Lovastatin (Calbiochem) was dissolved in absolute ethanol in 1 mM stockconcentration. Working concentration used in cell culture experiment was1 μM.

NP 101 formulation: NP101 was prepared by mixing 10 μM ATO, 10 μMCholecalciferol and 1 μM Lovastatin into a single mix by mixing itimmediately with osteogenic media (OM).

qRT-PCR: Total RNA was isolated first with Trizol (Invitrogen) and thenwith RNeasy Kit (Qiagen). First strand cDNA was generated using a Highcapacity cDNA reverse transcriptase kit (Abcam). qPCR was performedusing a Biorad cycler at 40 cycles of 95° C. for 5 seconds and at 60° C.for 30 seconds. PCR product was detected using SybrGreen (BioRad).Primers used for amplifications were Actin: Forward 5′-CAC AGC TTC TTTGCA GCT CCT-3′ (SEQ ID NO:1), Reverse 5′-CGT CAT CCA TGG CGA ACT G-3′(SEQ ID NO:2); Alk Phos: Forward 5′-CAC GCG ATG CAA CAC TCA GG-3′ (SEQID NO:3), Reverse 5′-GCA TGT CCC CGG GCT CAA AGA-3′ (SEQ ID NO:4).

Results

Relevant in vitro validations were performed for candidates like ArsenicTrioxide (ATO), Vitamin D and statins as single agents present duringosteogenesis and NP101 as a combination of agents for comparison. For afunctional assay an alkaline phosphatase (ALP) assay was used, as ALP isone of the very first enzymes active at the early stages ofosteogenesis. A key to understanding the role of ALP in mineralizationis provided by studies of the phased expression of genes duringosteoblastic differentiation and growth plate cartilage calcification.In both tissues, bone and calcifying cartilage, ALP must function earlyin the process of osteoblast formation. Osteoblasts are integral to theformation of heterotopic bone through production of alkaline phosphatase(AP).

Bone marrow stromal cells isolated from wild type mice and plated atfirst passage (P1) were used to test osteogenic properties in thepresence of ATO, cholecalciferol, lovastatin or NP101. An assay ofalkaline phosphatase, a widely recognized biochemical marker forosteoblast activity was utilized as a comparison tool. Alkalinephosphatase activity is clearly present in control BMSC cells, withoutany agent, after only four days in osteogenic media. Based on the numberof blue colored cells present in wells treated with OM and supplementedwith ATO, Cholecalciferol, lovastatin or NP101, it was found that thealkaline phosphatase activity decreases if agent is present (FIG. 1).

Similar results were obtained with respect to alkaline phosphatase geneexpression levels (Table 2). Compared to control (no agent present), allsingle agents an NP101 suppress alkaline phosphatase gene expressionsignificantly, and up to 90%.

TABLE 2 The expression of a differentiation marker, Alkaline Phosphatasein BMSC culture after four days supplementation of OM media without(control) agents present, with single agents or NP101 calculated using ΔΔ Ct method Control (no agent ATO, Cholecalciferol, Lovastatin, NP 101,NP 101, present) 10 μm 10 μm 1 μm 2× diluted 10× diluted ALP gene 1.000.105 ± 0.01 0.350 ± 0.02 0.316 ± 0.02 0.156 ± 0.02 0.500 ± 0.075expression fold change

The effect of NP101 was explored in cells primed for osteogenesis, andhaving a higher expression profile of bone related markers than control.Recent study suggests that BMSC cells pretreated with Lacritin 2 (LCN2)and Prolactin (PRL) express higher mRNA levels of bone related markersand calcium deposits than control (cells without pretreatment). BMSCcells were primed for osteogenesis and then exposed to osteogenic mediasupplemented with single or combined agents (FIG. 2). Treatment withsingle agents, like ATO, Cholecalciferol or lovastatin clearly diminishALP activity in cells, while NP101 even in its diluted form has the mostprominent effect on reducing alkaline phosphatase activity, based onpresence of bluish/purple cells.

All references cited herein are fully incorporated by reference. Havingnow fully described the invention, it will be understood by one of skillin the art that the invention may be performed within a wide andequivalent range of conditions, parameters and the like, withoutaffecting the spirit or scope of the invention or any embodimentthereof.

What is claimed is:
 1. A method for preventing or treating heterotopicossification, vascular calcification, or other pathologic calcificationin a patient, comprising administering to said patient a combination of:a) a Hedgehog (Hh) pathway antagonist; and b) vitamin D, cholecalciferolor a vitamin D analog, wherein the combination of said Hh pathwayantagonist and said vitamin D, cholecalciferol or vitamin D analog iseffective at preventing or treating said heterotopic ossification,vascular calcification, or other pathologic calcification in saidpatient.
 2. The method of claim 1, wherein said method is used toprevent heterotopic ossification in said patient, and wherein said Hhpathway antagonist and said vitamin D, cholecalciferol or vitamin Danalog are administered in a co-timely manner.
 3. The method of claim 2,wherein said Hh) pathway antagonist is administered at 1-500 mg/day; andsaid vitamin D, cholecalciferol or a vitamin D analog is administered at300-3000 IU/day.
 4. The method of claim 1, wherein said method is usedto prevent vascular calcification or other pathologic calcification insaid patient, and wherein said Hh pathway antagonist and said vitamin D,cholecalciferol or vitamin D analog are administered in a co-timelymanner.
 5. The method of claim 4, wherein said Hh) pathway antagonist isadministered at 1-500 mg/day; and said vitamin D, cholecalciferol or avitamin D analog is administered at 300-3000 IU/day.
 6. The method ofclaim 1, wherein said method is used to treat heterotopic ossificationin said patient, and wherein said Hh pathway antagonist and said vitaminD, cholecalciferol or vitamin D analog are administered in a co-timelymanner.
 7. The method of claim 6, wherein said Hh) pathway antagonist isadministered at 1-500 mg/day; and said vitamin D, cholecalciferol or avitamin D analog is administered at 300-3000 IU/day.
 8. The method ofclaim 1, wherein said method is used to treat vascular calcification orother pathologic calcification in said patient, and wherein said Hhpathway antagonist and said vitamin D, cholecalciferol or vitamin Danalog are administered in a co-timely manner.
 9. The method of claim 8,wherein said Hh) pathway antagonist is administered at 1-500 mg/day; andsaid vitamin D, cholecalciferol or a vitamin D analog is administered at300-3000 IU/day.
 10. A method for preventing or treating heterotopicossification, vascular calcification, or other pathologic calcification,in a patient, comprising administering to said patient a combination of:a) a Hedgehog (Hh) pathway antagonist; and b) a statin, wherein thecombination of said Hh pathway antagonist and said statin is effectiveat preventing or treating said heterotopic ossification, vascularcalcification, or other pathologic calcification in said patient. 11.The method of claim 10, wherein said method is used to preventheterotopic ossification in said patient, and wherein said Hh pathwayantagonist and said statin are administered in a co-timely manner. 12.The method of claim 11, wherein said Hh) pathway antagonist isadministered at 1-500 mg/day and said statin is administered at 1-500mg/day.
 13. The method of claim 10, wherein said method is used toprevent vascular calcification or other pathologic calcification in saidpatient, and wherein said Hh pathway antagonist and said statin areadministered in a co-timely manner.
 14. The method of claim 13, whereinsaid Hh) pathway antagonist is administered at 1-500 mg/day and saidstatin is administered at 1-500 mg/day.
 15. The method of claim 11,wherein said method is used to treat heterotopic ossification in saidpatient and wherein said Hh pathway antagonist and said statin areadministered in a co-timely manner.
 16. The method of claim 15, whereinsaid Hh) pathway antagonist is administered at 1-500 mg/day and saidstatin is administered at 1-500 mg/day.
 17. The method of claim 12,wherein said method is used to treat vascular calcification or otherpathologic calcification in said patient, and wherein said Hh pathwayantagonist and said statin are administered in a co-timely manner. 18.The method of claim 17, wherein said Hh) pathway antagonist isadministered at 1-500 mg/day; and said vitamin D, cholecalciferol or avitamin D analog is administered at 300-3000 IU/day.
 19. The method ofclaim 13, further comprising administering to said patient vitamin D,cholecalciferol or a vitamin D analog.
 20. The method of claim 14,wherein said Hh pathway antagonist and said statin are administered in asingle unit dosage form.
 21. The method of claim 15, and wherein: a)said Hh pathway antagonist is selected from the group consisting of: a)zerumbone epoxide; b) staurosporinone; c) 6-hydroxystauro-sporinone; d)arcyriaflavin C; e) 5,6-dihyroxyarcyriaflavin A; f) physalin F; g)physalin B; h) cyclopamine; i) HPI-1, HPI-2; HPI-3; or HPI-4; j) arsenictrioxide (ATO); k) sodium arsenite; 1) phenylarsine; m) GANT-58; n)GANT-61; o) zerumbone; and p) inhibitors of the expression of the genesPtch1, Gli1 or HIP; and/or b) said statin is selected from the groupconsisting of: Atorvastatin; Fluvastatin; Pravastatin; Rosuvastatin;Simvastatin; Pitavastatin; Cerivastatin; Lovastatin; and Mevastatin.